KR20050019075A - Injectable solid hyaluronic acid carriers for delivery of osteogenic proteins - Google Patents

Abstract

Injectable or implantable rod formulations for delivery of osteogenic proteins are disclosed. Formulations include hyaluronic acid derivatives and osteogenic proteins and optional excipients and active ingredients such as bone resorption inhibitors. Also disclosed are methods of making injectable rodable pharmaceutical compositions and methods of using osteogenic compositions for treating osteoporotic and / or osteopenic bone.

Description

INJECTABLE SOLID HYALURONIC ACID CARRIERS FOR DELIVERY OF OSTEOGENIC PROTEINS}

The present invention relates to the field of osteogenic proteins and pharmaceutical formulations thereof. More particularly, the present invention involves injectable or implantable solid pharmaceutical formulations containing hyaluronic acid derivatives and osteogenic proteins.

Idiopathic osteoporosis is an unknown etiology characterized by progressive bone mass loss and increasing vulnerability resulting in significant fracture sensitivity. Osteoporosis is one of the most prevalent of all musculoskeletal disorders and affects 56% of women over 45 years old (Praemer et al., Musculoskeletal Conditions in the United States, American Academy of Orthopaedic Surgeons, Park Ridge, IL (1992)). As the incidence increases with age and the proportion of older people in the population is increasing, osteoporosis will become more and more common in the future. Osteoporosis is difficult to treat locally, and there are currently no known therapies. Finally and most importantly, osteoporosis is associated with significant morbidity and mortality. The most serious fracture caused by osteoporosis is the fracture of the proximal femur in the hip region. With an incidence of more than 300,000 people annually, hip fractures are currently the most common fractures in older people. One in every six white women will have hip fractures throughout their lives (Cummings et al., Arch Intern Med 149: 2455-2458 (1989)), and one in three will reach 90 years of age.

Approximately 20% of those who live independently at the time of hip fracture remain in the long-term care facility for more than a year after the fracture. During the first year after injury, mortality is approximately 15% higher than age and gender matched controls [Praemer et al., Supra]. The increased incidence of proximal femur fractures observed in older patients is mainly associated with increasing tendency to fall as well as decreasing bone density of their proximal femurs. There is an inverse relationship between age-related bone loss in the proximal femur and the risk of hip fracture. A decrease of 1 standard deviation (SD) 1 in femoral neck bone density increased the risk of age-controlled hip fracture by 2.6-fold, respectively (95% Cl 1.9-3.6), with women in the lower 1/4 of the bone density grouping in the upper 1 / The risk of hip fractures is 8.5 times greater than women belonging to [4] (Cummings et al., The Lancet 341: 72-75 (1993)). The correlation between hip bone mass and hip fracture risk allows for screening and identification of patients for fracture risk. Patients with 2 standard deviations lower than the peak hip bone mass pass below the “fracture threshold”.

Current treatments for osteoporosis are systemic. These include fluoride, bisphosphonates, calcitonin, estrogens and progestins, testosterone, vitamin D metabolites, and / or calcium. In the United States, only estrogens, alendronates, and bisphosphonates have been suggested for preventing hip fractures in postmenopausal osteoporotic women. Each of these formulations requires continuous administration over several years.

In addition to the treatment of osteoporotic bone, there is a need for methods of treating or preventing osteoporosis-related fractures, for example by topical administration of osteogenic proteins. Because of this need, there is a need for a safe, effective and generally applicable carrier for topical treatment of bone defects despite the variety and availability of carrier materials for delivering osteogenic proteins. Thus, despite considerable efforts in the art, there remains a need for effective methods of repairing and / or treating osteoporotic and osteopenic bone and minimizing or reducing the incidence or severity of osteoporosis-related fractures.

Summary of the Invention

The present invention provides an injectable or implantable solid rod-like composition for intraosseous delivery of bone morphogenic proteins. In one embodiment, the composition contains osteogenic protein and hyaluronic acid ester. In another embodiment, the composition may further contain a bone resorption inhibitor such as bisphosphonate. In another embodiment, the composition may further contain one or more excipients, such as pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, or surfactants. The solid rod injectable or implantable compositions of the present invention prolong the retention of osteoinducing agents at the site of administration.

The present invention further provides methods and compositions for increasing bone mass and quality and for minimizing or reducing the incidence or severity of osteoporosis-related fractures. Accordingly, the present invention provides methods and compositions useful for reducing the incidence of fractures in osteoporotic or osteopenic bone. In particular, the present invention includes methods of treating patients with osteoporosis or other evidence of osteoporosis or osteopenic conditions. Preferred embodiments in which the present invention may prove particularly useful include the treatment of proximal femurs (hips), proximal humerus (hips), distal radius (wrist), and vertebral bodies (vertebrae), particularly interosseous bones including vertebral bodies. do.

The method includes an effective amount of at least one active agent at an osteopenic or osteoporotic bone site, or at a site of low bone mass or density, that can induce bone growth, increase bone tissue formation, or reduce bone loss at the site. Administering a containing solid bar composition. Bone mass is commonly referred to as "bone mineral content" or "BMC" and is measured in grams. Bone density is commonly referred to as "bone mineral density" or "BMD" and is expressed in grams per unit area or grams per unit volume. In certain embodiments, the mode of administration is by intraosseous injection. In the illustrated embodiment, the active agent is one or more proteins selected from the group of proteins known as the transforming growth factor-beta ("TGF-β") superfamily of proteins, as well as other proteins described in more detail herein. Preferably, bone morphogenic protein ("BMP"), growth and differentiation factor ("GDF"). The methods and compositions of the present invention are beneficial in that they provide localized treatment for osteoporosis or osteopenic bone but not systemic treatment. The present invention is also advantageous in that it utilizes, as active agents, bone morphogenic proteins that can be produced through recombinant DNA technology, which can potentially be supplied indefinitely. The methods and compositions of the present invention are also beneficial in that regeneration of bone tissue increases bone mass / density and increases bone strength, thereby reducing the incidence of osteoporosis or osteoporotic lesions and ultimately reducing the incidence of bone fractures. Do.

In another embodiment, the active agent is also added to one or more accessory proteins, such as Hedgehog, Noggin, Chordin, Frazzled, Cerberus and Follistatin, soluble BMP receptors, or in addition to one or more proteins selected from the TGF-β superfamily of proteins. It contains other proteins or agents as described.

The invention also administers an injectable rod-like composition containing one or more osteogenic proteins and a second composition containing an effective amount of an inhibitor of bone resorption, thereby increasing bone mass and quality and minimizing the incidence or severity of osteoporosis related fractures. Or a method for reducing. The second composition containing the bone reuptake inhibitor may be administered before, after or substantially simultaneously with the bone forming composition.

In addition to the healing of osteoporotic bone, the compositions of the present invention are injectable formulations of BMPs for use as injections into joints, for example, to promote cartilage repair and to treat and repair bone defects, cartilage defects and inhibit cartilage degradation. May be useful for Formulations may also be injected into the site of attachment to the tendons, ligaments and / or their bones. Injectable formulations of BMP can also be applied to other bone sites, such as bone sacs, implants into bone, closed or open fractures, and expanded bone formation.

In certain embodiments, the compositions of the present invention are prepared by a method comprising mixing the osteogenic protein and the hyaluronic acid derivative to form a osteogenic mixture. The bone forming mixture is then extruded and dried, for example, into air or a nonsolvent such as ethanol to form the bone forming mixture. The compositions of the present invention may further contain bone resorption inhibitors and / or excipients, either or both of which may be included in the mixing step.

Hyaluronic acid derivatives may be natural or synthetic hyaluronic acid, or modifications thereof. Hyaluronic acid is a naturally occurring polysaccharide comprising alternating N-acetyl-D-glucosamine, and D-glucuronic acid monosaccharide units linked by beta 1-4 bonds and disaccharide units linked by beta 1-3 glycoside bonds. It is usually produced as a sodium salt and has a molecular weight ranging from about 50,000 to 8 x 10 ⁶ . Bone resorption inhibitors include bisphosphonates such as alendronate, simandronate, clathronate, EB-1053, etidronate, ibandronate, nerridronate, olpadronate, pamideronate, risedronate, tilide Ronate, YH 529, zoledronate, and pharmaceutically acceptable salts, esters, acids and mixtures thereof. Excipients can be agents which control and / or stabilize the release of the active ingredient (s) such as pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers and / or surfactants. The osteogenic protein may be in solid or liquid form and the hyaluronic acid derivative and excipient (s) may be in solid form. Molding can be accomplished by extruding the bone forming mixture into air or nonsolvents such as ethanol; Drying can be accomplished by air drying or freeze drying. Sustained release preparations may also contain bone resorption inhibitors such as bisphosphonates.

The present invention also admixes bone forming proteins and hyaluronic acid or hyaluronan-based materials to form admixtures, compresses the admixtures to form a dense bone forming admixture, and then dense bone forming admixtures into the body. Provided is a method of making an injectable sustained release formulation comprising forming into a solid cylindrical rod suitable for injection or implantation into a rod. The forming step may be performed by forming a cylindrical rod through extrusion, compression, molding, drilling and / or cutting. Injectable sustained release preparations may further contain bone resorption inhibitors such as bisphosphonates, and one or more excipients such as, for example, those described above.

Injectable solid, bone inducible compositions of the invention may have a diameter of about 0.1 to 3.0 mm, preferably about 0.5 to 1.5 mm. The length of the solid rod-shaped composition may be about 1 mm to about 10 cm, in particular about 2 cm to about 5 cm. The compositions of the present invention may have a height to diameter ratio in the range of about 1000: 1 to 1: 1. The ratio is about 1000: 1, 500: 1, 250: 1, 100: 1, 50: 1, 25: 1, 10: 1, 5: 1, 4: 1, 3: 1, 2: 1 or 1: Can be 1 The osteoinductive composition of the present invention is typically rigid (but not broken) to withstand loading into a needle or syringe and injection into an intraosseous site. The osteoinductive composition has a density of about 0.5 to 100% material, preferably about 50 to 90% material. The composition has a low macroporosity and a low water content of about 0.1 to about 10.0% water, in particular about 0.1 to about 5% water. The ratio of active ingredient to carrier may be about 0.01-0.90 g active ingredient for about 1 g of carrier, in particular 0.1-0.3 g active ingredient for about 1 g of carrier. The hyaluronic acid derivative may be a partial or complete ester comprising about 50 to about 100% hyaluronic acid esterification.

Brief description of the drawings

1 is a graph showing local retention time of BMP-2 for various hyaluronic acid / rhBMP-2 compositions in a rabbit distal femur (intraosseous) model.

Description of the implementation

According to the present invention there is provided a method and composition for the treatment of a patient who exhibits signs of osteoporosis, or osteopenic condition, including osteoporotic bone lesions. Identification of the patient can be accomplished by procedures well known in the art. The procedure includes dual-energy X-ray absorption (DEXA) [Kilgus et al., J. Bone & Joint Surgery, 75-B: 279-287 (1992); Markel et al., Acta Orthop. Scand. 61: 487-498 (1990); And quantitative computed tomography (QCT) [Laval-Jeantet et al., J. Comput. Assist. Tomogr., 17: 915-921 (1993); Markel, Calcif. Tissue Int. 49: 427-432 (1991); Single photon absorption method [Markel et al., Calcif. Tissue Int. 48: 392-399 (1991); Ultrasonic transmission rate (UTV) [Heaney et al., JAMA 261: 2986-2990 (1989); Langton et al., Clin. Phys. Physio. Meas. 11: 243-249 (1990); And radiation evaluation [Gluer et al., J. Bone Min. Res. 9: 671-677 (1994)]. Other methods of patient identification for bone fracture risk include age-related factors such as recognition as well as assessment of previous occurrences of osteoporosis-related fractures [Porter et al., BMJ 301: 638-641 (1990); Hui et al., J. Clin. Invest. 81: 1804-1809 (1988). Said document is incorporated herein by reference.

The method includes injecting at least one purified osteogenic protein effective in inducing bone formation and / or maintenance, and a solid rod-like composition containing hyaluronic acid as a carrier to an osteoporotic or osteopenic site. Unlike conventional injectable formulations, the osteogenic compositions of the invention are administered in solid form to avoid defects inherent in liquid or viscous formulations. For example, using liquid or gel formulations, the osteoinducer may be prediluted with body fluids before the bone promoting effect is achieved. The present invention avoids the dilution effect by using a solid carrier that degrades slowly in vivo to provide prolonged and delayed release of the active ingredient (s). In addition, unlike liquid or viscous formulations that can migrate from the site of administration, the solid compositions of the present invention remain fixed at the site of bone growth desired to exhibit bone growth promoting activity. Typically, the composition should be maintained at this site for about 7 days to about 6 months. If the composition is predispersed, the desired bone growth promoting effect will not occur or the bone formed will not have the desired strength. Finally, the osteogenic composition of the present invention is administered as a solid, but it is formed into a cylindrical rod that is suitable for injection or implantation into the body. In addition, well-known surgical complications that induce embolism during intraosseous injection surgery are significantly alleviated through the use of solid rods (versus liquid or gel). Since the injection volume of the highly concentrated solid rod carrier is much smaller than the volume required when the similar dose is dispensed in liquid or gel form, there is a potential for intraosseous bone fragments, fat displacement or embolism caused by pressurized injection of a large volume of liquid / gel carrier. Is reduced. The composition can be applied to the desired bone growth site in any convenient manner, including introduction via conventional hypodermic needles or syringes.

The composition of the present invention is prepared by mixing a bone forming protein, hyaluronic acid carrier and optional excipients to form a viscous liquid / gel or paste depending on the hyaluronic acid starting material. The material obtained is then shaped into a cylindrical rod and dried. Shaping can be performed using any one of several known techniques, for example by molding, compression, drilling and / or cutting. In a preferred embodiment, the osteogenic mixture is packed and extruded through the center end of the hypodermic syringe. The material is extruded into a continuous cylindrical rod, dried at room temperature and segmented into small, injectable rods.

Hyaluronic acid starting materials are hydrophobic solids such as Hyaff-11 In the case of hyaluronic acid is first solubilized in an organic solvent to form a solution. The organic solvent may be any pharmaceutically acceptable solvent such as N-methylpyrrolidone (NMP) or dimethyl sulfoxide (DMSO), preferably NMP. The solution may contain about 1 to about 50% (w / v) hyaluronic acid, preferably about 5 to 20% (w / v), most preferably about 10% (w / v) hyaluronic acid. The dry powdered bone morphogenetic protein is dispersed in a hyaluronic acid solution at a concentration of about 1 to about 50% (w / w), preferably about 20% (w / w), and optionally excipients (eg, amino acids, sugars, salts, Surfactants, polymers, etc.) are added at a concentration of about 1 to about 50% (w / w), preferably about 20 to about 40% (w / w). The composition of the present invention may further contain an inhibitor of bone resorption, which may be included in the mixing step, either individually or in combination with bone forming protein components, in dry powder or in soluble form.

Hyaluronic acid starting material is in a hydrophilic form, such as Hyaff-11p65 If, hyaluronic acid can be combined with an aqueous buffer containing any excipient until the material has a paste density. The paste-like material contains about 1 to about 40% (w / v), preferably about 5 to about 30% (w / v), more preferably about 15 to about 20% (w / v) hyaluronic acid. can do. In the illustrated embodiment, the paste-like material contains 18.75% (w / v) hyaluronic acid. The dry powdered osteogenic protein is then mixed into the hyaluronic acid paste prior to shaping. Alternatively, in addition to blending with an aqueous buffer containing optional excipients, the hyaluronic acid starting material may be combined with an aqueous buffer containing soluble osteogenic protein with optional excipients until the material has a pasty density or viscous liquid or gel appearance. Can be combined. The composition may further contain an inhibitor of bone resorption, which may be included in the mixing step, either individually or in combination with the osteogenic protein component, in dry powder or in soluble form.

Once the components have been combined and formulated into a paste or viscous liquid or gel, the bone forming material can be a cylindrical mold, air or gas permeable tube (eg silicone rubber or Teflon). / FEP), or into an extruded equipment such as a syringe. When a syringe is used in the forming step, a plunger of the syringe is inserted and a sufficient amount of pressure is applied so that in the case of water soluble hyaluronic acid the continuous length paste is extruded onto the dry surface. In the case of water-insoluble hyaluronic acid, a continuous length of gel is extruded into a nonsolvent capable of precipitating the material. The segment is then cut using a cutting tool such as a laser, a scalpel, a knife, or the like to form an injectable rod-like composition. After segmentation, the rod-like composition is dried, for example by air drying or lyophilization.

The invention also provides a solid suitable for admixing bone forming proteins, hyaluronic acid or hyaluronan based materials, and optional excipients to form a dense osteogenic admixture and then injecting or implanting the dense osteogenic admixture into the body. Provided is a method of making an injectable sustained release formulation, comprising forming into a cylindrical rod.

The active agent can be selected from a family of proteins known as the transforming growth factor-beta (TGF-β) superfamily, including activin, inhibin, and bone morphogenic protein (BMP). In particular, active agents include one or more proteins selected from a subclass of proteins commonly known as BMPs, which are disclosed to have osteogenic activity and other growth and differentiation type activities. Such BMPs include, for example, US Pat. Nos. 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; And BMP proteins BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7 which are disclosed in 5,141,905; BMP-8 disclosed in PCT Publication WO91 / 18098; And BMP-9 disclosed in PCT Publication WO93 / 00432, BMP-10 disclosed in PCT Publication WO94 / 26893; BMP-11 disclosed in PCT Application WO94 / 26892, or BMP-12 or BMP-13 disclosed in PCT Application WO95 / 16035; BMP-15, disclosed in US Pat. No. 5,635,372; Or BMP-16 disclosed in US Pat. No. 6,331,612. Other TGF-β proteins that may be useful as active agents in the present invention include Vgr-2 [Jones et al., Mol. Endocrinol. 6: 1961-1968 (1992), and PCT application WO94 / 15965; WO94 / 15949; WO95 / 01801; WO95 / 01802; WO94 / 21681; WO94 / 15966; WO95 / 10539; WO96 / 01845; Any growth and differentiation factor (GDF) is included, including those described in WO96 / 02559 and elsewhere. Also disclosed herein are BIPs disclosed in WO94 / 01557; HP00269 disclosed in JP Publication No. 7-250688; And MP52 disclosed in PCT application WO93 / 16099. The disclosures of all such applications are incorporated herein by reference. Preferred subsets of BMPs for use in the present invention include BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12 and BMP-13. In the illustrated embodiment, the active agent is BMP-2, the sequence of which is disclosed in US Pat. No. 5,013,649, the disclosure of which is incorporated herein by reference. Other BMP and TGF-β proteins known in the art can also be used.

Active agents can be produced recombinantly or purified from protein compositions. The active agent may be homodimeric if it is a TGF-β, such as BMP, or other dimeric protein, or may be heterologous consisting of another BMP and (eg, one monomer of each of BMP-2 and BMP-6) Heteromers) or other members of the TGF-β superfamily such as activin, inhibin and TGF-β1 and (eg, a heterodimer consisting of one monomer of the relevant member of each BMP and TGF-β superfamily) It may be dimeric. Examples of such heterodimeric proteins are described, for example, in published PCT patent application WO93 / 09229, which is incorporated herein by reference.

The active agent may further contain additional agents such as Hedgehog, Frazzled, Chordin, Noggin, Cerberus and Follistatin proteins. The protein family is generally described in Sasai et al., Cell, 79: 779-790 (1994) (Chordin); PCT Patent Publication WO94 / 05800 (Noggin); And Fukui et al., Dev. Biol. 159: 131-139 (1993) (Follistatin). Hedgehog proteins are described in WO96 / 16668; WO96 / 17924; And WO95 / 18856. The Frazzled protein family is a relatively recently discovered protein family with high homology to the extracellular binding domain of the receptor protein family known as Frizzled. Genes and proteins of the Frizzled family are described by Wang et al., J. Biol. Chem. 271: 4468-4476 (1996). Active agents may also include other soluble receptors, such as the cleaved soluble receptors disclosed in PCT patent publication WO95 / 07982. In accordance with the teachings of WO95 / 07982, those skilled in the art will recognize that cleaved soluble receptors can be prepared from several different receptor proteins. These will also be included in the present invention. This publication is incorporated herein by reference.

The amount of active agent useful herein is an amount effective to stimulate the increased osteogenic activity of the present or infiltrating progenitor or other cells and will be based on the size and nature of the defect being treated. In general, the amount of protein to be delivered ranges from about 0.1 to about 500 mg, in particular from about 10 to about 300 mg, more particularly from about 150 to about 250 mg per cm 3 of material required.

Substances that may be useful as carriers in the practice of the present invention, when mixed and dried with a bone morphogenic protein, form a composition that possesses suitable handling characteristics for injectable or implantable application to osteoporotic or osteopenic bone sites. Pharmaceutically acceptable materials that have sufficient rigidity are included. The incorporation of bone morphogenic proteins in solid carriers increases the natural rate in other cases of regenerative osteogenic activity of infiltrating mammalian progenitor cells or other cells, and provides space for new tissue to grow and allow cell growth. Allow the protein to remain at the disease or lesion site for a time sufficient to form. The carrier also allows the bone morphogenic protein to be released from the disease or lesion site over an appropriate time interval to optimally increase the rate of regenerative osteogenic activity of progenitor cells. The carrier can also supply a skeleton that induces new formation in severe osteoporotic bone.

In the illustrated embodiment, the carrier family includes hyaluronic acid ester or hyaluronan based materials. As used herein, the terms "hyaluronic acid", "hyaluronan based material" and "hyaluronic acid derivative" are used interchangeably to form hyaluronic acid (as defined below) and salts thereof such as sodium, potassium, magnesium, Calcium salts; After molding and drying, the hyaluronic acid carrier is in a form suitable for injection or implantation, such as a cylindrical rod as described in detail below. The rod is hard enough to withstand injection into the intraosseous space as well as loading into conventional hypodermic needles or syringes. Hyaluronic acid based carrier materials are hard but have high tensile strength and low fractureability. The solid rod composition of the present invention preferably has a relatively high material density (30-100%), low macroporosity and low water content.

Hyaluronic acid is naturally produced in many tissues including lubricating fluids, vitreous fluids, human umbilical cords and chicken crest. It is the main component of the intracellular matrix of connective tissues such as skin, tendons, muscles and cartilage. In addition to providing mechanical support for the tissue cells, hyaluronic acid also promotes other important biological functions including hydration, lubrication, cell migration and differentiation (eg Balazs et al., Cosmetics & Toiletries 5 (84): 8-). 17). Hyaluronic acid can be extracted from natural tissue, such as chicken rice, or produced by recombinant methods. The molecular weight of hyaluronic acid obtained by extraction is generally in the range of 800 to 13 million. Polysaccharides are unstable and easily degraded by various physical (mechanical, radioactive) and chemical agents. As a result, conventional purification procedures generally produce hydrolyzed fractions of low molecular weight hyaluronic acid (see Balazs et al., Supra).

The term "hyaluronic acid" as used herein refers to acidic polysaccharides comprising D-glucuronic acid and N-acetyl-D-glucosamine residues, including various molecular weight fractions and mixtures of derivatives thereof, regardless of molecular weight. . Derivatives of hyaluronic acid include, for example, hyaluronic acid chemically modified through esterification, crosslinking, sulfated, and the like. Hyaluronic acid is an ester, for example Jeanloz et al., J. Biol. Chem. 186: 495-511 (1950); Jager et al., J. Bacteriology 1065-1067; Biochem. J. 167: 711-716 (1977); Jeanloz et al., J. Biol. Chem. 194: 141-150 (1952); Or methyl ester of hyaluronic acid as described by Jeanloz et al., Helvetica Chimica Acta 35: 262-271 (1952).

In the illustrated embodiment, hyaluronic acid is an ester of an aliphatic, aromatic, aroaliphatic, cycloaliphatic or heterocyclic alcohol with hyaluronic acid, all or a portion of the carboxyl groups of the acid esterified, such as hereby incorporated by reference in its entirety. Hyaluronic acid derivatives described in US Pat. No. 5,336,767. The hyaluronic acid starting material may be as described in co-pending US application Ser. No. 09 / 687,283, filed Oct. 13, 2000, which is hereby incorporated by reference in its entirety. In particular, hyaluronan-based starting materials are solids such as nonwoven pads, felts, sheets, powders, sponges and the tradename Hyaff under Fidia Advanced Biopolymers (Abano Terme, Italy). Commercially available microspheres. Hyaff Materials are described, for example, in US Pat. No. 4,851,521, which is incorporated by reference in its entirety; 4,965,353; And 5,202,431; And EP 0 216 453. Hyaff Substances of hyaluronic acid with varying degrees of esterification (ie partial esters or full esters) as well as one or combined ester moieties (such as benzyl, ethyl, propyl, pentyl or larger molecules such as hydrocortisone or methyl prednisone) Ester. Hyaff Partial esters of the substance are represented by% esterification in the range of 50-99% (eg Hyaff-11p65 And Hyaff-11p80 ), The complete ester is a 100% ester of hyaluronic acid (eg, Hyaff-11 ). In addition to providing the desired handling features for the compositions of the present invention, Hyaff The materials also provide a means for biocompatibility and absorption dynamics manipulation of the active ingredient (s) [eg, US Pat. No. 6,339,074, which is incorporated herein by reference in its entirety; 6,232,303; And 6,066,340].

In another exemplary embodiment, the hyaluronan based starting material is a nonwoven fabric comprising natural polymers, semisynthetic derivatives of natural polymers and / or mixtures of synthetic polymers and hyaluronic acid ester fibers. The mixture may contain about 1 to about 100% hyaluronic acid. Natural polymers useful in the present invention include, without limitation, collagen or co-precipitates of collagen and glycosaminoglycans; cellulose; Polysaccharides such as chitin, chitosan, pectin or pectic acid, agar, agarose, xanthan gum, gellan, alginic acid or alginate, polymannan or polyglycans, starch and natural rubber. Semi-synthetic derivatives of natural polymers useful in the present invention include, for example, aldehydes or aldehyde precursors, dicarboxylic acids or their halides, diamines, cellulose derivatives, alginic acid, starch, hyaluronic acid, chitin or chitosan, gellan, xanthan, pectin or Natural polymers such as collagen cross-linked with agents such as pectic acid, polyglycans, polymannans, agar, agarose, natural rubber and glycosaminoglycans. Synthetic polymers include, for example, polylactic acid, polyglycolic acid, polydioxane, polyphosphazene, polysulfone resins, polyurethane resins and copolymers and derivatives thereof. Examples of nonwoven fabric materials useful in the present invention, including methods for their preparation, are described in US Pat. No. 5,520,916, issued May 28, 1996, which is incorporated herein by reference in its entirety.

While much is known about the osteogenic potential of TGF-β protein, recent reports indicate that topical administration of certain osteoinducing agents, such as BMP-2, promotes transient osteolytic activity at the site of administration. This reaction, followed by new bone formation induced by BMP, was termed "temporary resorption phenomenon".

Agents known to inhibit bone resorption may play an important role in delaying or reducing initial bone resorption associated with local BMP administration without inhibiting subsequent bone formation. Clinically, bisphosphonate therapy has been shown to significantly reduce bone turnover, increase bone mineral density, and reduce hip and spinal fracture risk in osteopenic women (eg, Fleisch, which is incorporated by reference in its entirety herein). , H., Bisphosphonates In Bone Disease, From The Laboratory To The Patient, section 3, Parthenon Publishing (1997). That is, in one embodiment, a bone reuptake inhibitor, such as bisphosphonate, is co-administered with an osteoinducer to prevent or minimize initial bone reuptake associated with intraosseous delivery of BMP. Coadministration of bisphosphonates leaves the bone reinforcing effect still occurring while blocking the undesirable resorption phase.

Despite their therapeutic efficacy, bisphosphonates have poor absorption in the gastrointestinal tract when taken orally. In order to overcome the poor bioavailability problem, intravenous administration was used; However, this aspect is expensive and appears to be inconvenient due to the duration and frequency of administration. The present invention overcomes these disadvantages by incorporating bisphosphonates into the carrier and delivering them directly to the desired site of action.

In one embodiment of the invention, the bone reuptake inhibitor is incorporated into an injectable osteoinductive composition as the second active ingredient. Bone reuptake inhibitors may be mixed with osteogenic proteins, hyaluronic acid carriers, and / or any excipient (s) prior to the shaping and drying step. The final mixture is then shaped, for example, by extrusion into a nonsolvent or air.

In another embodiment of the invention, the bone resorption inhibitor is administered sequentially or simultaneously with the bone forming composition. According to this embodiment, the osteogenic composition can be delivered locally to specific areas in need of bone growth or repair, with simultaneous or subsequent administration of a bone reuptake inhibitor in a separate delivery vehicle. That is, the bone reuptake inhibitor may be injected or implanted directly into the site to be treated, for example by injection or surgical implantation in a sustained release carrier. The carrier may be any pharmaceutically acceptable carrier, a wide range of which is well known and readily available in the art (e.g., Martin, EW, Remington's Pharmaceutical Sciences (Mack, which is incorporated herein by reference in its entirety). Pub. Co., recent edition). Preferably the carrier is a sustained release carrier, most preferably hyaluronic acid ester or a hyaluronan based material as described above. Presently preferred carriers are formed of solid rods as described elsewhere herein.

The term "bone resorption inhibitor" as used herein refers to the prevention of bone loss through direct or indirect modification of osteoclast formation or activity, in particular inhibition of existing bone removal. That is, the term "bone resorption inhibitor" as used herein refers to an agent that prevents or inhibits bone loss by direct or indirect modification of osteoclast formation or activity.

As used herein, the term “bisphosphonate” refers to the related bisphosphonic acids and salts, and to various crystalline and amorphous forms of bisphosphonates. In certain embodiments, the bisphosphonates can be alendronates, simdronates, clathronates, EB-1053, etidronates, ibandronates, nerridronates, olpadronates, pamideronates, risedronates, tilides Ronate, YH 529, zoledronate, and pharmaceutically acceptable salts, esters, acids and mixtures thereof.

The amount of bone reuptake inhibitor useful herein is an amount effective to prevent or inhibit the initial bone loss typically associated with topical administration of BMP by direct or indirect modification of osteoclast formation or activity. The exact required dosage will be based on the amount of bone forming agent delivered as well as the size and nature of the defect being treated. Generally, the amount of phosphonate to be delivered is in the range of about 1 to about 3000 mg, in particular about 10 to about 1000 mg, and illustratively about 100 to about 500 mg per cm 3 of material. The site of application is preferably topical (intraosseous) but may be other parenteral sites for systemic delivery, such as intramuscular or subcutaneous.

Additional additives or excipients that may be useful in the compositions of the present invention include, without limitation, pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and / or surfactants. Such excipients for stabilizing and / or controlling the active ingredient (s) are well known in the formulation art. Useful polymers include, for example, those described in US Pat. No. 5,171,579, which is incorporated herein by reference in its entirety. Synthetic polymers or surfactants include, but are not limited to, pluronic, such as Poloxamer 407 gels, a class of water-soluble ABA type block surfactant copolymers that exhibit the unique properties of reversed phase gelatin. Other useful synthetic polymers include poly (lactide) / poly (ethylene glycol), polyvinylpyrrolidone (PVP), poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly (vinyl alcohol) Polylactide and polyethylene glycol. Natural polymers include, without limitation, sodium alginate, chitosan, collagen, gelatin, hyaluronan, and cellulosic materials such as hydroxycellulose. Other useful excipients include peptides, proteins, and amino acids.

In one embodiment of the invention, the excipients are presented in powder form and then solubilized Hyaff-11 in an organic solvent with the active agent (s). It is mixed into and extruded into ethanol (non-solvent) to form a rod which is then rinsed and dried. The final composition may contain one or a combination of excipients, preferably salts, sugars (eg sucrose) and / or amino acids (eg glycine and / or glutamic acid). Compositions of the present invention may contain about 1 to about 60% (w / w) amino acids, about 1 to about 60% (w / w) sugar, and about 1 to about 60% (w / w) synthetic polymers. . In another embodiment of the invention, the formulation is about 20-50% (w / w) amino acid, and / or about 5-50% (w / w) sugar, and / or about 20-50% (w / w) contains a synthetic polymer.

Injectable compositions of the invention may be administered by any clinically acceptable injection mode. Several commercial syringes are suitable for use in the present invention and for administering the compositions of the present invention. For example, suitable syringes are commercially available Calasept containing sterile calcium hydroxide paste in isotonic saline solution in a non-aspirated or modified inhalation cartridge syringe. Syringe [JS Dental Manufacturing, Ridgefield CT]; Henke-Ject Suction Syringe and Hypo Dental syringes / needles [Smith & Nephew MPL, Franklin Park, IL]; Intraosseous needle [MPL, Inc., Chicago IL]; And Luer-Lok Syringes [Becton Dickinson, Franklin Lakes, NJ], all of which may be suitable syringes for use in the present invention. Any syringe that can hold and deliver the injectable rod and / or can be extruded into the closure is suitable for use.

In one embodiment of the invention, the solid rod-like composition is delivered into the bone using a hypodermic needle of the appropriate size and type subcutaneously or surgically pre-positioned into a selected anatomical position. Transdermal displacement of the hypodermic needle can be achieved using hand palpation of known anatomical indicators, with or without fluoroscopy to visualize the displacement. Fluoroscopy may also be used with surgical implants before and / or at the same time as displacement of the hypodermic needle.

In the illustrated embodiment, the guide line (commonly referred to as "k-line") is first inserted transdermally into the desired anatomical position to serve as a guide for the hypodermic needle. The transdermal needle is inserted over the guide line that is removed after leaving the hypodermic needle in place. The solid rod-like composition is then inserted into the central tip of the hypodermic needle. After loading of the composition, a second guide line is inserted into the needle and used to push the solid composition to the needle tip. The needle is then removed after the guide has secured the composition in the bone at the desired location. Finally, the guideline is removed leaving the solid composition in place. In another embodiment, the solid rod-like composition of the present invention is prepositioned in the needle barrel. After displacement into the desired anatomical site, the plunger of the syringe is pushed into the needle barrel while withdrawing the device, leaving the solid rod-like composition in the desired position.

In one embodiment of the invention, the bone morphogenic protein is used as an osteoinductive agent for treating osteoporosis. Patients who may benefit from such treatment include bone mass using dual-energy X-ray absorption (DEXA), quantitative computed tomography (QCT), single photon absorption, ultrasound transmission rate (UTV), and / or radiographic assessment. / Can be identified using any one or more of a variety of standard procedures, including density measurements. The procedure provides the physician with information about the location and severity of osteoporotic or osteopenic bone lesions. In addition to locating the lesion (s) to be treated, the physician can use this information to select the appropriate mode of administration and dose of osteoinducing agent to the patient.

In another embodiment of the invention, the bone morphogenic protein is used as an osteoinductive agent in a method known as expanded bone formation. The method is an alternative to segmented bone regeneration for the implanted osteoinducing agent. In traditional segmented bone repair, osteoinducing agents and carriers are located in defects created between parent bone ends. For bone formation to occur, the osteoinducing agent must have sufficient retention time in the defect to promote the differentiation of a sufficient number of osteogenic cells to support new bone formation. The expanded bone formation method produces regenerating constructs between the expanded parental bone ends that contain a large population of blood vessels and a large population of mesenchymal stem cells that have been determined to become osteogenic cells. As a result, regenerative constructs represent a much more ideal environment in which cell differentiation growth factors, such as rhBMP-2, promote rapid bone induction compared to bone induction in segmental defects.

The dilated bone formation procedure begins with an initial latent phase that allows fibrous connections to form between the bone ends to be expanded. Following this latent phase, the bone end expands slowly at a controlled rate of up to 1 mm per day in the human clinic. Once the regen has been formed and the bone end has been extended to an appropriate length, an extended reinforcement is needed to allow the regen to form a bone. The extended enhancers, which can be on the scale of 4 to 6 months, are associated with significant morbidity. Frequent complications are the occurrence of pin area infections caused by extended time of the external fixator used to keep the extension in place. In addition, there are significant psychological effects and lifestyle changes associated with wearing external fixators over extended periods of time. In addition to complications associated with external anchorages, there are several patients in which the regeneration is not properly formed and the union is prolonged or the union is not formed. Since the regeneration includes reactive cell populations and already abundant blood vessels following the initial expansion phase, the use of bone morphogenic proteins can rapidly accelerate bone formation rates during the generally extended reinforcement phase of expansion bone formation. Acceleration in expansion is limited to the relaxation of the soft tissue associated with the bone. Cells generated using expanded bone formation can also be harvested to provide a primed cell source for bone formation. The cells can be cultured to produce immortalized cell lines. If desired, the cells can also be immunotolerated using agents such as CTLA4 receptor [US Pat. No. 5,434,131] or CTLA4 ligand or B7 monoclonal antibody [WO96 / 40915]. Methods and materials for such immunotolerance are disclosed in the above references and include cotransfection or treatment with such factors. The disclosure of this reference is incorporated herein by reference.

In addition to the treatment of osteoporosis and closed fractures, the rod-like compositions of the present invention can also be applied to other bone sites such as bone sacs and defects. Injectable solid compositions may also be administered into non-bone sites, such as tendons, damaged cartilage tissue, ligaments and / or their attachment sites to the bone.

Although the above discussion is directed to the administration of a single osteoinductive composition, the present invention naturally includes co-administration of multiple active ingredients in individual formulations, for example the bisphosphonate compositions described above. Multiple active ingredients can be delivered simultaneously or sequentially, individually or in combination in separate delivery vehicles.

Dosage regimens will be determined by various patient variables (such as body weight, age, gender) and clinical symptoms (such as extent of injury, site of injury, etc.) as well as the clinical symptoms present.

The compositions of the present invention allow a therapeutically effective amount of osteoinductive protein to be delivered to the site of injury where cartilage and / or bone formation is desired. Formulations are in the orthopedic field; Cranial / chin face reconstruction; For bone regeneration in osteomyelitis; And for autologous bone grafts, spinal fusion, and bone defect repair in fresh nonunion fractures in the dental field for alveolar augmentation and periodontal defects and ankles. When used to treat osteomyelitis or to repair bone with minimal infection, the osteogenic protein can be used in combination with antibiotics. Antibiotics are selected for their ability to have minimal adverse effects on bone formation while reducing infection. Preferred antibiotics for use in the compositions of the present invention include vancomycin and gentamicin. The antibiotic can be in any pharmaceutically acceptable form, such as vancomycin HCl or gentamicin sulfate. The antibiotic is preferably present at a concentration of about 0.1 mg / ml to about 10.0 mg / ml. Traditional preparations of formulations in pharmaceutically acceptable form (ie, pyrogen free, appropriate pH and isotonicity, sterility, etc.) are well known in the art and applicable to the formulations of the invention.

The solid rod-like compositions of the invention can also be used in combination with other drugs, growth factors, peptides, proteins, cytokines, oligonucleotides, antisense oligonucleotides, DNA and polymers. The compounds can be added by mixing them with the hyaluronic acid carrier or covalently attaching to the carrier. The hyaluronic acid composition can also be used with cells transduced or transfected with DNA encoding BMP and genes encoding BMP protein.

The following examples illustrate the invention and do not limit it in any way. Modifications, modifications and minor enhancements are included and are within the invention.

Example 1 Formulation of Hyaff-11 Rods

Injectable 100% esterified Hyaff-11 rod-shaped compositions (1 mm in diameter) were prepared and evaluated for retention and bone formation efficiency of recombinant human bone morphogenic protein-2 (rhBMP-2). The rod-like composition contains Hyaff-11 as a carrier The hyaluronan base material, two doses of rhBMP-2 as active ingredient (see Table 1), and various amounts of excipients were included for controlling the release kinetics. The excipients used in this example were in the form of a dry powder of glutamic acid or buffer salts. The buffer salt contained 0.5% sucrose, 2.5% glycine, 5 mM L-glutamic acid, 5 mM NaCl, and 0.01% polysorbate 80. Hyaff-11 The base composition was formed into a rod using a phase inversion method. Briefly, rhBMP-2 and excipients (glutamate and buffer salts) are Hyaff-11 in pre-solubilized organic solvent N-methylpyrrolidone (NMP). The particles were mixed into (10% w / v) and extruded into excess ethanol (non-solvent) using a syringe and catheter (eg 16-gauge), phase reversed for 1 hour, rinsed and dried. The drying step consisted of 24 hours air drying followed by 24 hours freeze drying. Extrusion was carried out using a metered syringe pump, preferably at an injection rate of 0.2 ml / min. Hyaff-11 The base composition was prepared: Hyaff-11 , 20% (w / w) rhBMP-2, and 40% (w / w) glutamate (ie 40/40/20 (w / w) Hyaff-11 / Glutamate / rhBMP-2); Hyaff-11 , 60% (w / w) rhBMP-2 / bufferitis (ie 40/60 (w / w) Hyaff-11 / rhBMP-2); And Hyaff-11 , 20% (w / w) rhBMP-2, and 20% (w / w) buffer salt (ie, 60/20/20 (w / w) Hyaff-11 / Bufferitis / rhBMP-2). High dose rhBMP-2 was obtained by desalting the protein formulation prior to combination with Hyaff-11. For further evaluation, the dry bar was typically cut into 1 or 2 cm segments. Theoretical capacity of the bars is listed in Table 1. A preferred mode of administration is a 16-gauge subcutaneous needle equipped with a closure for injecting the solid intramembrane into the intraosseous site.

Injectable Hyaff RhBMP-2 dosage for rod formulation Theoretical capacity Formulation (BMP-2 μg / rod mg) (BMP-2 mg / rod cm) 40/40/20 (w / w) Hyaff-11 / Glutamate / rhBMP-2 200 1.5 40/60 (w / w) Hyaff-11 / rhBMP-2 71 0.5 60/20/20 (w / w) Hyaff-11 / Bufferitis / rhBMP-2 200 1.3 80/20 (w / w) Hyaff-11p65 / rhBMP-2 200 1.2 60/40 (w / w) Hyaff-11p65 / rhBMP-2 400 2.4

Example 2: Hyaff-11p65 Formulation of the rod

Injectable 65% esterified Hyaff-11p65 rod-like compositions (1 mm in diameter) were prepared and evaluated for retention and bone formation efficiency of rhBMP-2. The rod-like composition contains Hyaff-11p65 as a carrier. Hyaluronan based material and 2 doses of rhBMP-2 as active ingredient (see Table 1) were contained. Desalted rhBMP-2 and Hyaff-11p65 in dry form After mixing the nonwoven pad, it is hydrated to 18.75% (w / v) of the nonwoven pad weight, mixed to a white pasty density, delivered by syringe, extruded through a catheter (eg 16-gauge) and dried, 20% (w / w) rhBMP-2 (ie 80/20 (w / w) Hyaff-11p65 / rhBMP-2) or 40% (w / w) rhBMP-2 (ie 60/40 (w / w) Hyaff-11p65 / rhBMP-2) containing Hyaff-11p65 The base composition was prepared. A variant of the method consists of extruding the paste into a rod through a catheter, freezing the rod (in -80 ° C or liquid nitrogen), inserting into a slightly larger diameter tube (such as a 14-gauge catheter) and drying it. lost. The drying step consisted of 24 hours air drying followed by 24 hours freeze drying. Alternative methods of rod manufacture include Hyaff-11p65 1.5mm inner diameter silicone rubber paste or Teflon Molding into a / FeP tube and then drying. Preferred mode of administration is a 16-gauge subcutaneous needle equipped with a closure for inserting the solid intramembrane into the intraosseous site.

Example 3: In Vitro Analysis

All rod-like compositions were solid, straight, handleable, and injectable through 16-gauge needles. Hyaff-11 Scanning electron microscopy (SEM) of the rod composition was typically solid, dense and smooth, while Hyaff-11p65 The rod composition was densely packed into short fibrous segments of natural nonwoven fibers. The bioactivity of rhBMP-2 in the rod-like composition was obtained by extracting rhBMP-2 from the composition and evaluating its ability to induce alkaline phosphatase (bone marker) expression in mouse W-20-17 stromal cells. Hyaff-11 And Hyaff-11p65 RhBMP-2 from the rod composition was bioactive.

Example 4: Local Biodistribution in vivo

Hyaff-11 And Hyaff-11p65 The rod composition was used to obtain a range of rhBMP-2 retention profiles in vivo. Local retention times of rhBMP-2 in 1 cm rod compositions (prepared as described in Examples 1 and 2) were evaluated in a rabbit distal femur bone model using ¹²⁵ I-rhBMP-2 and gamma scintillation measurements ( 1). Hyaff-11 (Hyaff-11p65 Formulations) provided slow and sustained rhBMP-2 release regardless of the BMP-2 dose or excipient. Sterilization of glutamate excipients with ethylene oxide provided slightly more sudden release during the initial three day period compared to gamma-sterilized glutamate. 80/20 (w / w) Hyaff-11p65 The / rhBMP-2 composition gave the fastest release kinetics of rhBMP-2.

Example 5: Efficacy and Biocompatibility

Hyaff-11 prepared as described above Base composition (Hyaff-11p65 Not) were evaluated for effects and biocompatibility on bone formation two weeks after subcutaneous (dorsal thoracic) and intraosseous (distal femur) administration in rats. The rod-like compositions for intraosseous and subcutaneous administration were 2 mm and 10 mm long, respectively. Radiography and histological analysis of the subcutaneous administration site show bone formation near the rod-shaped composition containing rhBMP-2, resulting in rhBMP-2 / Hyaff-11 It was suggested that the rod was osteoinductive (data not shown). Both subcutaneous and intraosseous administration sites showed minimal inflammatory response, suggesting biocompatibility of the hyaluronic acid / BMP-2 composition. Hyaff-11 And Hyaff-11p65 The rod composition is additionally injected into the rabbit distal femur, in particular 80/20 (w / w) Hyaff-11p65 / rhBMP-2 and 40/40/20 (w / w) Hyaff-11 In the / glutamate / rhBMP-2 formulation, significant new bone formation was observed histologically within the intraosseous space after 7 weeks (data not shown). 80/20 (w / w) Hyaff-11p65 into the distal radius of ovarian abdominal baboon Injection of the / rhBMP-2 rod formulation gave a 30% relative increase in columnar bone volume compared to histologically untreated control (data not shown).

Claims (71)

A composition for the injectable delivery of a bone morphogenic protein containing a bone morphogenic protein and a hyaluronic acid ester, the composition being in the form of a cylindrical rod suitable for injection or implantation in a solid state in the body. The composition of claim 1, wherein the osteogenic protein is selected from the group consisting of bone morphogenic protein (BMP) family members. The composition of claim 2 wherein the bone morphogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12, and BMP-13. The composition of claim 2, wherein the osteogenic protein is BMP-2 or BMP-6. The composition of claim 1 further comprising an inhibitor of bone resorption. 6. The composition of claim 5, wherein the bone resorption inhibitor is bisphosphonate. 7. The bisphosphonate of claim 6, wherein the bisphosphonate is an alendronate, a shimadronate, a clathronate, an EB-1053, an etrideronate, an ibandronate, a neridronate, an olpadronate, a pamideronate, a risedronate, a tilide. Ronate, YH 529, zoledronate, and pharmaceutically acceptable salts, esters, acids and mixtures thereof. The composition of claim 1 wherein the hyaluronic acid ester comprises about 50% to about 100% hyaluronic acid esterification. The composition of claim 1 wherein the hyaluronic acid ester is crosslinked hyaluronic acid. The composition of claim 1 further comprising an excipient selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants. The composition of claim 1 further comprising an excipient selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants, and inhibitors of bone resorption. The composition of claim 1, wherein the cylindrical rod has a diameter of about 0.5 to 1.5 mm. The composition of claim 1, wherein the cylindrical rod is about 2 cm to 5 cm in length. A composition for treating osteoporotic bone, containing osteogenic protein and hyaluronic acid, prepared by a method comprising: (a) mixing the osteogenic protein with the hyaluronic acid ester to form a osteogenic mixture; And (b) forming and drying the osteogenic mixture into a cylindrical rod suitable for injection or implantation into the body in a solid state. 15. The composition of claim 14, wherein the mixing step comprises mixing the bone morphogenic protein and hyaluronic acid ester with bisphosphonate. The excipient according to claim 14, wherein in the mixing step, an excipient selected from the group consisting of bone forming proteins and hyaluronic acid esters and pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants Compositions Including Mixing. 15. The excipient according to claim 14, wherein the mixing step comprises bone forming proteins and hyaluronic acid esters, and excipients selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants, And a mixture of bisphosphates. 15. The composition of claim 14, wherein the hyaluronic acid ester is prepared by hydration or solubilization of insoluble or partially soluble particles, films, fibers, nonwoven pads, or sponges of hyaluronic acid benzyl ester in water, organic solvents or aqueous buffers. 15. The excipient according to claim 14, wherein the mixing step comprises bone forming proteins and hyaluronic acid esters, and excipients selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants, And a mixture of bisphosphates. The composition of claim 14, wherein the bone morphogenic protein is selected from the group consisting of bone morphogenic protein (BMP) family members. 15. The composition of claim 14, wherein the osteogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12, and BMP-13. The composition of claim 14, wherein the osteogenic protein is BMP-2 or BMP-6. 16. The bisphosphonate of claim 15, wherein the bisphosphonate is an alendronate, a simandronate, a clathronate, an EB-1053, an etrideronate, an ibandronate, a neridronate, an olpadronate, a pamideronate, a risedronate, a tilide. Ronate, YH 529, zoledronate, and pharmaceutically acceptable salts, esters, acids and mixtures thereof. 15. The method of claim 14, wherein the step of mixing comprises mixing the bone forming protein and the hyaluronic acid ester with a solvent; Extrusion of the osteogenic mixture into the nonsolvent in the forming and drying step of the osteogenic mixture into a cylindrical rod. The composition of claim 18 wherein the forming and drying steps of the bone forming mixture comprise: (i) extruding the bone forming mixture into a nonsolvent, or (ii) extruding the bone forming mixture into air to dry. The composition of claim 25, wherein the nonsolvent is ethanol or water. 15. The composition of claim 14, wherein the cylindrical rod has a diameter of about 0.5 to 1.5 mm. 15. The composition of claim 14, wherein the cylindrical rod is about 2 cm to 5 cm in length. A process for preparing an injectable, sustained release formulation containing bone forming protein and hyaluronic acid ester, comprising: (a) a bone forming protein and hyaluronic acid ester, and an excipient selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants, about 1 to about Producing a bone forming mixture containing hyaluronic acid ester in an amount of about 50% (w / v); (b) shaping the bone forming mixture to form a rod-shaped product; And (c) drying the rod-like product from step (b). 30. The method of claim 29, wherein step (a) comprises solubilization of the hyaluronic acid ester into the organic solvent. 30. The method of claim 29, wherein step (a) comprises hydration of the hyaluronic acid ester into the aqueous buffer. The method of claim 29, wherein the hyaluronic acid ester in step (a) is about 10 to about 25% (w / v). The method of claim 29, wherein the forming in step (b) comprises: (i) extruding the bone forming mixture in a nonsolvent, or (ii) extruding the bone forming mixture into air to dry. The method of claim 29, wherein the bone morphogenic protein is selected from the group consisting of bone morphogenic protein (BMP) family members. 33. The method of claim 32, wherein the osteogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12, and BMP-13. 33. The method of claim 32, wherein the osteogenic protein is BMP-2 or BMP-6. 30. The method of claim 29, wherein the mixing in step (b) further comprises mixing bone forming proteins, hyaluronic acid esters, and excipients with bisphosphates. 36. The bisphosphonate of claim 35, wherein the bisphosphonate is an alendronate, a simandronate, a clathronate, an EB-1053, an etrideronate, an ibandronate, a neridronate, an olpadronate, a pamideronate, a risedronate, a tilide. Ronate, YH 529, zoledronate, and pharmaceutically acceptable salts, esters, acids and mixtures thereof. 30. The method of claim 29, wherein the hyaluronic acid ester comprises about 50% to about 100% hyaluronic acid esterification. A method of making an injectable sustained release formulation containing a bone forming protein and a hyaluronan based material, comprising: (a) admixing the bone morphogenic protein and the hyaluronic acid based material to form an admixture; (b) compacting the admixture from step (a) to form a dense bone forming admixture; And (c) forming the dense osteogenic admixture from step (b) into a solid cylindrical rod suitable for injection or implantation into the body. 39. The method of claim 38, wherein said forming during step (c) consists of extrusion, compression, molding, drilling, or cutting to form a cylindrical rod of 0.5 to about 1.5 mm diameter. 39. The method of claim 38, wherein the admixture of step (a) comprises a bone forming protein and a hyaluronan-based material and a pharmaceutically acceptable salt, polysaccharide, peptide, protein, amino acid, synthetic polymer, natural polymer, and surfactant. And admixture of excipients selected from the group consisting of. 39. The method of claim 38, wherein the admixing during step (a) comprises admixing the bone forming protein and the hyaluronan based material with bisphosphonates. 40. The method of claim 39, wherein the osteogenic protein is selected from the group consisting of bone morphogenic protein (BMP) family members. The method of claim 38, wherein the osteogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12, and BMP-13. The method of claim 38, wherein the osteogenic protein is BMP-2 or BMP-6. 42. The bisphosphonate of claim 41, wherein the bisphosphonate is an alendronate, a simandronate, a clathronate, an EB-1053, an etrideronate, an ibandronate, a neridronate, an olpadronate, a pamideronate, a risedronate, a tilide. Ronate, YH 529, zoledronate, and pharmaceutically acceptable salts, esters, acids and mixtures thereof. The method of claim 38, wherein the hyaluronan base material is an ester comprising from about 50% to about 100% hyaluronic acid esterification. A method for treating a mammal with a bone defect comprising administering an effective amount of the bone forming composition to a bone defect site, wherein the bone forming composition contains a bone forming protein and a hyaluronic acid ester, the composition being in the form of a cylindrical rod Way. 48. The method of claim 47, wherein the bone defect is osteoporotic or osteopenic bone. 48. The method of claim 47, wherein the osteogenic protein is selected from the group consisting of bone morphogenic protein (BMP) family members. The method of claim 49, wherein the bone morphogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12, and BMP-13. The method of claim 49, wherein the osteogenic protein is BMP-2 or BMP-6. 48. The method of claim 47, further comprising an inhibitor of bone resorption. The method of claim 52, wherein the bone resorption inhibitor is bisphosphonate. 55. The bisphosphonate of claim 53, wherein the bisphosphonate is an alendronate, a simandronate, a clathronate, an EB-1053, an etrideronate, an ibandronate, a neridronate, an olpadronate, a pamideronate, a risedronate, a tilide. Ronate, YH 529, zoledronate, and pharmaceutically acceptable salts, esters, acids and mixtures thereof. 48. The method of claim 47, wherein the hyaluronic acid ester is an ester comprising from about 50% to about 100% hyaluronic acid esterification. 48. The method of claim 47, further comprising an excipient selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants. 48. The method of claim 47, further comprising an excipient selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants, and inhibitors of bone resorption. A method of treating a mammal having a bone defect, comprising: (a) administering to the bone defect site an effective amount of a bone forming composition containing bone forming protein and hyaluronic acid ester and in the form of a cylindrical rod; And (b) administering an effective amount of a bone resorption inhibitor to the bone defect site. 59. The method of claim 58, wherein the bone defect is osteoporotic or osteopenic bone. 59. The method of claim 58, wherein the osteogenic protein is selected from the group consisting of bone morphogenic protein (BMP) family members. 59. The method of claim 58, wherein the osteogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12, and BMP-13. 59. The method of claim 58, wherein the osteogenic protein is BMP-2 or BMP-6. 59. The method of claim 58, wherein the bone resorption inhibitor is bisphosphonate. 64. The bisphosphonate of claim 63, wherein the bisphosphonate is an alendronate, a simandronate, a clathronate, an EB-1053, an etrideronate, an ibandronate, a neridronate, an olpadronate, a pamideronate, a risedronate, a tilide. Ronate, YH 529, zoledronate, and pharmaceutically acceptable salts, esters, acids and mixtures thereof. 59. The method of claim 58, wherein the hyaluronic acid ester comprises about 50% to about 100% hyaluronic acid esterification. 59. The method of claim 58, wherein the bone forming composition further comprises an excipient selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants. 59. The method of claim 58, wherein step (a) is performed before step (b). 59. The method of claim 58, wherein step (b) is performed before step (a). 59. The method of claim 58, wherein step (a) is performed substantially simultaneously with step (b).